Mycobacterium tuberculosis, the causative agent of tuberculosis, is an opportunistic pathogen associated with HIV. A 50% higher rate of incidence is seen in HIV positive individuals as compared to HIV negative individuals. Antibiotic regimens can decrease the number of living bacteria, but are unable to eliminate the bacteria. New drugs are needed that are capable of keeping the pathogen from emerging from the host or drugs that can clear the individual of the bacteria. The manzamine family of alkaloids has shown promising activity against M. tuberculosis. All but one of the manzamine family members inhibits 98-99% Mtb with MIC <12.5 pg/mL. Manzamine A and 8-hydroxy manzamine A have both been shown to inhibit Mtb protein kinases, PknE and PknL, involved in cell division and replication of the bacteria. Toxicity from the P-carboline moiety in the molecule is responsible for the narrow therapeutic window and limited use as a drug. Modeling suggests that this group intercalates DNA disrupting topoisomerase interactions, and it is also capable of stabilizing a carbocation. Both the hydrophobic heterocycle, ircinal A, and the [unreadable]-carboline are involved in the inhibition. Ircinal A represents an outstanding scaffold for building manzamine analogs with the [unreadable]-carboline replaced with other heterocycles that maintain important interactions, and therefore lower the chance of toxicity. Ircinal A is a low abundance product that is impossible to synthesize efficiently. Because this molecule is an intermediate or shunt product of the manzamine biosynthetic pathway, identification of this pathway from Micromonospora sp. M42 can lead to controlled production of ircinal A. Isolation and identification of the genes responsible for manzamine A production will involve molecular biological and biochemical techniques. Feeding studies involving isotopically labeled precursors will be used to help establish proposed biosynthetic pathways. Degenerate primers based on these pathways will be used to help identify potential genes of the production. Mutants that lose the ability to produce manzamine A will be generated in a traceable method. The expression profile of M42 under producing and non-producing conditions will be analyzed by comparison of isotope incorporation into protein. Knowledge of the biosynthetic genes will allow for ircinal production and also production of Mtb drug candidates. PUBLIC HEALTH RELEVANCE: "Tuberculosis is a major disease among HIV positive patients and new treatments are needed. One promising lead is manzamine. By contolling the production of this compound and related compounds better drugs can be made in a rational way."